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Fungi colonize diverse ecologic niches, yet their cellulases are based on a central theme — exo- and endo-splitting glucanases acting co-operatively and in some instances, synergistically. The characterization of cellulases has proved to be complex. This is due in part to the ill-defined nature of the natural substrates, plus the multiple nature of cellulase components. The rationales for this multiplicity continues to be explored. Cloning studies have shown far less diversity at the genomic level. Additionally, the endo- and exo-splitting glucanases have been shown to be comprised af a tripartite structure with both enzyme forms containing a cellulose binding site, hinge region and catalytic domain. Oxidative enzymes (cellobios quinone oxido-reductase, cellobiose oxidase and lactonase) though minor in quantitative terms, may have important roles in the regulation/initiation of cellulase synthesis per se.

A protein present on the cell surface of Bacillus licheniformis (Bl) NM105 was identified as an S-layer (OlpA in this paper), a protein present on many bacterial cell surfaces. Purification, SDS-PAGE and isoelectrofocusing showed one 94-kDa, slightly acidic (pI 6.5) protein band (defined as OlpA). The pure protein OlpA, has a tetragonal symmetry of its morphological subunits. Following Edman degradation, three 17-mer oligodeoxyribonucleotide (oligo) probes corresponding to the N-terminal sequence of OlpA were synthesized and used for gene cloning. The nucleotide (nt) sequence of the cloned gene (olpA) showed an ORF and encoded an 874 amino acid (aa) protein. In the promoter region of olpA, there appear to be -10 and -35 ∂ A -binding sites, as well as -10 and -35 regions specific for∂ H . The existence of these two potential promoters suggests that OlpA would be produced during both the vegetative and sporulating stages of growth. The ribosome-binding site (RBS) sequence perfectly matched its consensus sequence, suggesting a high efficiency of translation of olpA. A typical 29-aa leader peptide, characteristic of secretory proteins in Bacilli , is present in the OlpA pre-protein sequence. In olpA, there are two stem-loop structures in tandem, downstream from the stop codon. These stem-loops are probably involved in prolonged olpA expression, by extending the half life of the mRNA.

A mutant of Bacillus licheniformis 749/C, NM 105 exhibits some notable properties, e.g., arrest of alkaline phosphatase secretion and overexpression and hypersecretion of RS protein. Although RS is known to be widely distributed in many microbes, it is rarely found, with a few exceptions, in laboratory cultures of microorganisms. RS protein is a structural protein and has the unusual properties to form aggregate. This characteristic may have been responsible for the self assembly of RS into regular tetragonal structures. Another uncommon characteristic of RS is that enhanced synthesis and secretion which occurs when the cells cease to grow. Assembled RS protein with a tetragonal structure is not seen inside cells at any stage of cell growth including cells in the stationary phase of growth. Gel electrophoresis of the culture supernatant shows a very large amount of RS protein in the stationary culture of the B. licheniformis. It seems, Therefore, that the RS protein is cotranslationally secreted and self assembled on the envelope surface.

Two strains of filamentous, branching, budding organisms were isolated, one from sand and the other from the foam of a sewage-treatment plant. These organisms are very fine, their hyphae and cells having a diameter of about 1 /um. They differ from actinomycetes in having a eucaryotic cytology, walls with no murein and being resistant to antibacterial antibiotics and sensitive to strictly antifungal ones. Considered as fungi they have been tentatively placed in the genus Fusidiumt Link. Unusual cytological features were observed. These included nuclei and mitochondria with membranous extensions, looped cristae in some mitochondria, an atypical nuclear membrane and an abundance of mesosomes.

Mycoplasma-1ike organisms (MLO) are pathogens affecting a large variety of plants. The MLOs infect mono- and dicotylidenous plants, ranging from large trees to cultivated crops and weeds. Mycoplasmas are wall-less pleomorphic, prokaryotic organisms extensively studied from mammalian sources. They are parasitic to animals and can be grown in pure culture, while MLOs from plant and insect sources have not yet been cultivated outside the hosts. Hence, no information on the physiology and genetics of MLOs are available. Some recent reports including a few papers presented in this volume indicate that the cultivation of MLOs might be routinely possible in the near future. When such material will become available, similarities and differences between plant MLOs and animal mycoplasmas will be studied in detail. The name MLO has been coined to indicate that not enough information is available to date to establish identity or differences between them and mammal-infecting mycoplasmas. The term “plant mycoplasmas” is often used to describe both MLOs and the culturable spiroplasmas.

Endoglucanase (EG) is an enzyme of cellulase complex depolymerizing crystalline cellulose to cellulose microfibril fragments. T. reesei Rut-C30 is a catabolite repression resistant mutant which enhances endoplasmic reticulum (ER) and produces 15-20 times more endoglycanase than the wild type QM6a. About 90% of this enzyme is secreted.Purified secreted EG from three day old culture was used for polyclonal antibody production. The IgG was labeled with 15-18nm colloidal gold. EG secreting mycellia, grown in Vogel's medium with 1% Avicel PH101, were collected by centrifugation. This pellate was washed in sodium cacodylate buffer, fixed in tannic acid-glutaraldehyde mixture and cryoprotected with 30% glycerol. This material was placed on the specimen holders and frozen in liquid freon followed by liquid nitrogen. Frozen thin sections were prepared in a LKB Cryonova microtome using glass knives and collected on sucrose drops.